[Effects of clomiphene on the functions of the corpus luteum].
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Clomiphene↗
Biomedical subjects
W Huang
Publications and source records attributed to W Huang.
[A study on the relationship between morphology and gene heterogeneity in acute promyelocytic leukemia].
Aucte promyelocytic leukemia (APL) can be treated by all-trans retinoic acid (ATRA) with high complete remission rate. 50 cases of APL diagnosed morphologically were studied on their cytogenetics, molecular biology and response to treatment with ATRA. Forty-five cases showed chromosomal translocation t(15; 17) and PML/RAR alpha fusion gene (PML + RAR alpha + APL). They had typical morphologic change, in which hypergranular cells appeared more frequently in L type of PML/RAR alpha and microgranular cells in S type of PML/RAR alpha. Among the 45 PML + RAR alpha + APL patients 8 died early and 37 had complete remission with ATRA. In the remainging 5 patients, three had typical APL morphologic features in cytology, but one of them displayed t (11; 17) with PLZF+RARA alpha +, the second showed RAR alpha + PML - (PML - RAR alpha + APL) and the third PML - RAR alpha -(PML - RAR alpha - APL). They did not respond to ATRA treatment. These data indicate that APL is not a homogeneous disease. The other a patients had neither chromosomal translocation nor rearrangements of the two genes. On careful morphological reexamination, these two cases were not APL, but one of them responded well to ATRA. It is shown that morphology is the important diagnostic basis of APL, but in a few APL cases diagnosis should be made with the help of cytogenetics and molecular biology. Response of ATRA treatment may be of diagnostic value of APL, but is not a specific criterion.
Adolescent↗
V-J junctional sequences of T cell receptor gamma gene in acute lymphocytic leukemia.
T cell receptor TCR gamma gene rearrangement in a series of acute lymphocytic leukemia (ALL) patients was studied using PCR technique. 18 V-J junctional sequences (designated by N sequence) of TCR gamma gene were amplified with the V gamma and J gamma primers, using an unsymmetrical PCR and analysed by direct sequencing, demonstrating that in Chinese ALLs, the N sequences of TCR gamma gene are indeed clone-specific. Based on the known N sequences, several oligo-nucleotides were synthesized as probes, which were specific for the leukemic clone, and were used to detect the minimal residual disease (MRD) in 4 ALL cases. The sensitivity of this method was 0.1%-0.01%.
Adult↗
Altered expressions of cardiac Na/K-ATPase isoforms in copper deficient rats.
OBJECTIVE: The aim was to determine if copper deficiency affects the expression of Na/K-ATPase alpha isoforms in the rat heart. METHODS: Copper deficiency was induced by placing weanling rats on a copper deficient diet for 4-5 weeks. Adult ventricular tissue, isolated ventricular myocytes, and brain stems of the control and deficient rats were compared for Cu, Zn-superoxide dismutase (CuZn-SOD) activity and for protein and mRNA contents of Na/K-ATPase alpha isoforms. RESULTS: In brain stem, where copper deficiency did not alter CuZn-SOD activity, mRNA and protein levels of alpha isoforms also remained unchanged. In ventricular tissue and ventricular myocytes, copper deficiency reduced CuZn-SOD activity, mRNAs of alpha 1 and alpha 2 isoforms, and the alpha 2 isoform protein. The alpha 1 isoform protein of ventricular tissue and its myocytes was marginally reduced by copper deficiency. CONCLUSIONS: In the rat ventricular tissue, oxidative stress resulting from copper deficiency (1) enhances the turnover of the more oxidant sensitive alpha 2 isoform to a greater extent than the turnover of the alpha 1 isoform; (2) regulates mRNA levels of alpha 1 and alpha 2 isoforms; and (3) contributes to the cardiomyopathy of copper deficiency.
Animals↗
[A suggestion for unification of present limb lead systems into a single "F" system].
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Adult↗
Natriuretic role of oxytocin in sodium homeostasis in rats.
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Animals↗
[Effects of Chinese herbs on impaired lymphocyte functions after thermal injury in mice].
Four Chinese herbs were chosen for this study. They were Panax pseudoginseng wall (PPW), Loniconica japonica thunb (LJT), Polygonum cuspidatum (PC) and Jilin ginseng (JG). They were given from the 2nd to the 7th postburn day (PBD). The results showed that the response to antigen signal, the proliferative capacity, the interleukin II production function, and the antibody production ability by lymphocytes in control burned mice were more noticeably reduced than that in the normal mice. The PPW, LJT, PC might restore all the impaired functions in different degrees. The JG showed no effect at all.
Animals↗
[Histochemical observation of the effect of electroacupuncture on the livers of rats with endotoxic shock].
The experimental rats were randomly divided into three groups: i.e. control group; endotoxic shock group (the model of endotoxic shock was induced by intravenous administration of E. Coli endotoxin, 16 mg/kg); and electroacupuncture (EA) group ("Renzhong" or "Zusanli" acupoint was stimulated for 15 minutes at 1 hr after injection of endotoxin). The experimental rats were decapitated at 75 minutes after injecting endotoxin. Their livers were taken out for cryostat section, and histochemical observation. The results were as follows: 1) The glycogen in the hepatic cells of endotoxic shocked rats were almost completely depleted. The activities of SDH Mg(++)-ATPase and G-6-Pase and 5'-Nase were decreased; especially the activities of 5'-Nase in the biliary canaliculi and sinusoids were apparently reduced. 2) The content of hepatic glycogen in EA group was increased, but some of them was still depleted. The activities of SDH, Mg(++)-ATPase and G-6-Pase were slightly increased as compared with that of the endotoxic shock group. The activity of 5'-Nase was obviously increased after EA. The preliminary results indicated that EA at "Renzhong" or "Zusanli" acupoint of rats with endotoxic shock might play certain role on improving the hepatic metabolism and promoting the membrane transport action.
Animals↗
Structure determination of aquomet porcine hemoglobin at 2.8 A resolution.
Considerable attention is currently focused on hemoglobins from lower mammals, such as the pig, for potential use in cell-free blood substitute preparations safe for use in humans. As the first step in the elucidation of structure-function relationships in porcine hemoglobin, we have determined the three-dimensional structure of aquomet porcine hemoglobin at 2.8 A resolution. Overall, the porcine hemoglobin tetramer is structurally similar to that of human oxyhemoglobin, and the r.m.s. deviation of all backbone atoms (minus five residues at the amino and carboxyl termini of each subunit) is 0.8 A. This similarity is not unexpected given that human and porcine hemoglobins exhibit 85% sequence identity. However, regions of subtle structural differences are implicated in subtle functional differences between the two proteins, such as the 20 to 25% inhibition of the alkaline Bohr effect and the accompanying reduction in oxygen-linked chloride binding observed for porcine hemoglobin. The structural similarity of these two mammalian hemoglobins also rationalizes the novel hybridization behavior of pig and human subunits in transgenic pigs expressing both porcine and human hemoglobins in porcine erythrocytes.
Amino Acid Sequence↗
Relationship between cytochrome P450 2E1 and acetone catabolism in rats as studied with diallyl sulfide as an inhibitor.
Previous studies have demonstrated that cytochrome P450 2E1 (P450 2E1) catalyzes the oxidation of acetone in vitro. The present study was designed to determine the importance of P450 2E1 in the catabolism of acetone in rats using diallyl sulfide (DAS) as an inhibitor of this enzyme. After a single intragastric dose of DAS, blood samples were collected from rats at different time points, and blood acetone concentrations were measured by gas chromatography. In a low DAS dose (50 mg/kg body weight) group, the maximum acetone level of 6-fold higher than the normal level was reached at 6 hr; the acetone level returned to normal at 48 hr. In a high dose (200 mg/kg) group, the maximum acetone level of 9-fold higher than the normal level was reached at 12 hr; the acetone level returned to normal at 60 hr. The turnover time and fractional turnover rate of elevated acetone were 15.8 +/- 0.5 hr and 0.054 +/- 0.001 hr-1, respectively, for the low dose, and 19.2 +/- 0.6 hr and 0.046 +/- 0.005 hr-1, respectively, for the high dose. In a chronic experiment, DAS (50 and 200 mg/kg, i.g.) was given to rats daily for 29 days, and elevated blood acetone levels were observed during the entire experimental period: 2.0 to 2.8 micrograms/mL for the low dose and 3.4 to 3.9 micrograms/mL for the high dose at 24 hr after the 1st, 7th, 14th and 28th doses versus 0.8 to 0.9 micrograms/mL for the control. The increase of blood acetone level was closely related to the decreases of N-nitrosodimethylamine (NDMA) demethylase activity and P450 2E1 content in liver microsomes. Consistent with the lack of cumulative effect from the multiple doses of DAS on acetone level, rather stable levels of the DAS metabolites, diallyl sulfoxide (45.0 micrograms/mL, range: 33.8 to 58.6 micrograms/mL) and diallyl sulfone (11.7 micrograms/mL, range: 6.9 to 15.6 micrograms/mL), were observed at 24 hr after the 1st, 7th, 21st and 28th doses with DAS (200 mg/kg) in the chronic experiment. It is likely that the inactivation and inhibition of P450 2E1 by DAS and its metabolites block the oxidation of acetone and cause its elevation in blood. The results strongly suggest an important role of P450 2E1 in acetone catabolism under physiological conditions.
Acetone↗
Isolation of a covalent steady-state intermediate in glutamate 60 mutants of thymidylate synthase.
Glutamate 60 of thymidylate synthase coordinates a hydrogen bond network important in proton transfer reactions to and from the substrate dUMP. The E60A and E60L mutants of Lactobacillus casei thymidylate synthase catalyzed tritium exchange from [5-3H]dUMP for solvent protons faster than dTMP formation, indicating accumulation of a steady-state intermediate and a change in partitioning of the intermediate. A covalent complex consisting of E60A or E60L thymidylate synthase, dUMP, and the cofactor CH2H4 folate was isolated on SDS-polyacrylamide gel electrophoresis and shown to be chemically and kinetically competent to form dTMP. These results provide proof of the formation of a covalent steady-state intermediate in the reaction pathway of thymidylate synthase and demonstrate that the rate-determining step in the mutants occurs during conversion of the covalent intermediate to dTMP.
Base Sequence↗
The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase.
Human plasma glutathione peroxidase (GSH-Px) is a distinct extracellular selenoenzyme that detoxifies hydroperoxides when used with GSH in high (mM) non-physiological concentrations. We have discovered that NADPH and human thioredoxin reductase (TR) by itself or with thioredoxin (Trx) are efficient electron donors to this human plasma peroxidase. Incubation of 0.05 microM TR with 0.25 microM GSH-Px, in a system free from GSH, resulted in reduction of t-butyl hydroperoxide. Addition of Trx, 2.5 and 5 microM, respectively, further increased the rate of the reaction. These data were obtained using an assay measuring the oxidation of NADPH. A direct assay demonstrated the formation of cumyl alcohol from cumene hydroperoxide in this GSH-independent peroxidase reaction. Incubation of 0.25 microM GSH-Px with a low concentration of GSH (10 microM), representing the upper level in plasma, plus excess glutathione reductase and NADPH did not result in any reduction of t-butyl hydroperoxide. However, after addition of 2.5 microM human glutaredoxin, a linear peroxidase reaction started. The results suggest that extracellular TR, Trx, or glutaredoxin are reductants for the selenium-dependent peroxidase rather than GSH.
Electron Transport↗
High-efficiency synthesis of human alpha-endorphin and magainin in the erythrocytes of transgenic mice: a production system for therapeutic peptides.
Chemical synthesis of peptides, though feasible, is hindered by considerations of cost, purity, and efficiency of synthesizing longer chains. Here we describe a transgenic system for producing peptides of therapeutic interest as fusion proteins at low cost and high purity. Transgenic hemoglobin expression technology using the locus control region was employed to produce fusion hemoglobins in the erythrocytes of mice. The fusion hemoglobin contains the desired peptides as an extension at the C end of human alpha-globin. A protein cleavage site is inserted between the C end of the alpha-globin chain and the N-terminal residue of the desired peptide. The peptide is recovered after cleavage of the fusion protein with enzymes that recognize this cleavage signal as their substrate. Due to the selective compartmentalization of hemoglobin in the erythrocytes, purification of the fusion hemoglobin is easy and efficient. Because of its compact and highly ordered structure, the internal sites of hemoglobin are resistant to protease digestion and the desired peptide is efficiently released and recovered. The applicability of this approach was established by producing a 16-mer alpha-endorphin peptide and a 26-mer magainin peptide in transgenic mice. Transgenic animals and their progeny expressing these fusion proteins remain health, even when the fusion protein is expressed at > 25% of the total hemoglobin in the erythrocytes. Additional applications and potential improvements of this methodology are discussed.
Amino Acid Sequence↗
Laminar comparison of somatosensory cortical plasticity.
During tactile learning there is a transformation in the way the primary somatosensory cortex integrates, represents, and distributes information from the skin. To define this transformation, the site of earliest modification has been identified in rat somatosensory cortex after a change in sensory experience. Afferent activity was manipulated by clipping all except two whiskers on one side of the snout ("whisker pairing"), and the receptive fields of neurons at different cortical depths were mapped 24 hours later. Neurons in layer IV, the target of the primary thalamic pathway, were unaltered, whereas neurons located above and below layer IV showed significant changes. These changes were similar to those that occur in layer IV after longer periods of whisker pairing. The findings support the hypothesis that the layers of cortex contribute differently to plasticity. Neurons in the supragranular and infragranular layers respond rapidly to changes in sensory experience and may contribute to subsequent modification in layer IV.
Action Potentials↗
Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime.
Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant. Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates. The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime. However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required. The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime. The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.
Anti-Bacterial Agents↗
Constitutive activation of Mek1 by mutation of serine phosphorylation sites.
A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.
Animals↗
Crystal structure of a catalytic antibody with a serine protease active site.
The three-dimensional structure of an unusually active hydrolytic antibody with a phosphonate transition state analog (hapten) bound to the active site has been solved to 2.5 A resolution. The antibody (17E8) catalyzes the hydrolysis of norleucine and methionine phenyl esters and is selective for amino acid esters that have the natural alpha-carbon L configuration. A plot of the pH-dependence of the antibody-catalyzed reaction is bell-shaped with an activity maximum at pH 9.5; experiments on mechanism lend support to the formation of a covalent acyl-antibody intermediate. The structural and kinetic data are complementary and support a hydrolytic mechanism for the antibody that is remarkably similar to that of the serine proteases. The antibody active site contains a Ser-His dyad structure proximal to the phosphorous atom of the bound hapten that resembles two of the three components of the Ser-His-Asp catalytic triad of serine proteases. The antibody active site also contains a Lys residue to stabilize oxyanion formation, and a hydrophobic binding pocket for specific substrate recognition of norleucine and methionine side chains. The structure identifies active site residues that mediate catalysis and suggests specific mutations that may improve the catalytic efficiency of the antibody. This high resolution structure of a catalytic antibody-hapten complex shows that antibodies can converge on active site structures that have arisen through natural enzyme evolution.
Amino Acid Sequence↗
Glucocorticoids potentiate the adenylyl cyclase-cAMP system mediated immunoreactive beta-endorphin production and secretion from hypothalamic neurons in culture.
Beta-endorphin(beta EP)1-31, a potent opioid peptide of proopiomelanocortin (POMC) derivatives, is produced and released from neurons at arcuate nuclei of the rat hypothalamus. Although dexamethasone (DM) suppresses the production and secretion of POMC related peptides from rat pituitary corticotrophs, the effect of glucocorticoids on the function of hypothalamic beta EP neurons remains unclear. Employing long term monolayer cultures of neonatal rat hypothalamic cells, we report here that 4 day treatment with 10 microM of forskolin increased ir-beta EP levels in cell content and culture media by approximately 1.7 (P < 0.05) and 4.1 times (P < 0.01) above vehicle treated control cultures (mean +/- S.E.M., 47.3 +/- 2.6 pg/well and 40.4 +/- 3.0 pg/well; n = 3) respectively. Although 4 day treatment with DM alone had little effect on the release and the cell content of ir-beta EP, it significantly enhanced forskolin-induced elevation of ir-beta EP levels in cell content and in culture media. The effect of DM was dose-related and time-dependent, with an EC50 of about 1 nM; at this concentration DM enhanced ir-beta EP secretion about 2.1 times (P < 0.01) above that induced by 10 microM of forskolin alone. Furthermore, the potentiating effect of DM was specifically suppressed by 100 nM of RU38486 (P < 0.01), a glucocorticoid receptor antagonist, but not by an equivalent dose of RU28318, a mineralocorticoid receptor antagonist. In addition, Northern blot analysis showed that forskolin (10 microM) increased the abundance of POMC mRNA 1.4 fold above that of vehicle treated control cultures. Whereas by itself, DM (10 nM) had little effect on the level of POMC mRNA, it enhanced forskolin-stimulated increase of the abundance of POMC mRNA approximately 2.6 times. Moreover, DM also augmented 1.6 times (P < 0.05) forskolin-induced but not 3-isobutyl-1-methylxanthine (IBMX)-induced increase of cAMP production (5.5 +/- 0.4 pmol/well; mean +/- S.E.M., n = 3) in the cultures. Taken together, our findings suggest that in contrast to the inhibitory effect on pituitary corticotrophs, glucocorticoids enhance the production and secretion of beta EP from rat hypothalamic neurons by facilitating the stimulatory effect mediated, in part, through the adenylyl cyclase-cAMP system.
1-Methyl-3-isobutylxanthine↗